The transcription factor NF-kappaB is regulated primarily through its association with the inhibitory protein IkappaB. The NF-kappaB/IkappaB complex is normally sequestered in the cytoplasm until cells are stimulated with an agonist such as interleukin-1 (IL-1), tumor necrosis factor (TNF), and lipopolysaccharides (LPS). Upon stimulation of cells, IkappaB is rapidly phosphorylated by an IkappaB kinase complex (IKK) and then degraded by the ubiquitin-proteasome pathway. The degradation of 1kappaB allows NF-kappaB to enter the nucleus to turn on downstream genes, many of which play pivotal roles in the life and death of cells. A key step in the NF-kappaB signaling pathway is the phosphorylation of IkappaB by the IKK complex. This kinase complex integrates signals from multiple pathways including those emanating from IL-1 and LPS. Genetics studies have demonstrated that TRAF6, a RING finger domain protein, and components of the IKK complex are essential for NF-kappaB activation in response to IL-1 and LPS. However, it is not known how TRAF6 activates IKK. Recent studies by the principal investigator's laboratory have shown that the activation of IKK complex by TRAF6 requires a dimeric ubiquitin conjugating enzyme complex, Ubc13/Uev1A, and the formation of a unique polyubiquitin chain linked through lysine-63 (K63) of ubiquitin. The goal of this proposal is to understand the novel mechanisms of ubiquitin-dependent activation of the IKK complex. Specifically, experiments are proposed to 1) study the structure and function of Ubcl3/Uev1A and TRAF6 as related to IKK activation; 2) identify and characterize additional factors required for IKK activation by TKAF6, including the ubiquitination target; 3) identify and characterize the K63-linked polyubiquitin chain binding protein; 4) investigate the mechanisms of ubiquitin-dependent activation of IKK by TRAF6. Taken together, these studies should fill significant gaps in the NF-kappaB signaling pathway, and provide the important pieces that together are required to solve the puzzle of IKB kinase activation by ubiquitin. Given the importance of NF-kappaB in human diseases, information gained from the proposed studies will be of direct relevance to biomedicine, including discovery of novel therapeutic targets.